Neuronal tracing of oral nerves in a velvet worm-Implications for the evolution of the ecdysozoan brain

Abstract

As one of the closest relatives of arthropods, Onychophora plays an important role in understanding the evolution of arthropod body plans. Currently there is controversy surrounding the evolution of the brain among the ecdysozoan clades, which shows a collar-shaped, circumoral organization in cycloneuralians but a ganglionic architecture in panarthropods. Based on the innervation pattern of lip papillae surrounding the mouth, the onychophoran brain has been interpreted as a circumoral ring, suggesting that this organization is an ancestral feature of Ecdysozoa. However, this interpretation is inconsistent with other published data. To explore the evolutionary origin of the onychophoran mouth and to shed light on the evolution of the ecdysozoan brains, we analyzed the innervation pattern and morphogenesis of the oral lip papillae in the onychophoran Euperipatoides rowelli using DNA labeling, immunocytochemistry, and neuronal tracing techniques. Our morphogenetic data revealed that the seven paired and one unpaired oral lip papillae arise from three anterior-most body segments. Retrograde fills show that only the first and the third nerves supplying the lip papillae are associated with cell bodies within the brain, whereas the second nerve exclusively receives fibers from somata of peripheral neurons located in the lip papillae. According to our anterograde fills and immunocytochemical data, the first nerve supplies the anterior-most pair of lip papillae, whereas the second and the third nerves are associated with the second to fifth and second to eighth lip papillae, respectively. These data suggest that the lip papillae of E. rowelli are mainly innervated by the proto- and deutocerebrum, whereas there are only a few additional cell bodies situated posterior to the brain. According to these findings, the overall innervation pattern of the oral lip papillae in E. rowelli is incompatible with the interpretation of the onychophoran brain as a modified circumoral ring.

Keywords: Cycloneuralia; Ecdysozoa; Onychophora; arthropods; central nervous system; lip papillae; mouth.

Figure 1

Phylogeny of Ecdysozoa (molting animals). Trichotomies indicate unresolved relationships. Phylogenetic relationships combined after Mayer and Whitington (2009b) and Nielsen (2012). Abbreviations: C, Cycloneuralia; E, Ecdysozoa; N, Nematoida; P, Panarthropoda; S, Scalidophora.

Figure 2

Diagrams illustrating segmental identity of modified cephalic appendages and position of corresponding neuronal somata in the central nervous system of Onychophora. Modified from Mayer et al. (2010). (A) Assignment of four anterior-most pairs of limbs to corresponding segments (demarcated by dotted lines). Black-filled circles indicate the position of eyes. (B) Position of neuronal cell bodies innervating the three pairs of cephalic appendages, including the antennae, the jaws, and the slime papillae. Note that the somata of neurons supplying the slime papillae lie outside the brain. Abbreviations: an, antennal nerve; at, antenna; br, brain; cp, connecting piece; dc; deutocerebral region; ey, eye; jn, jaw nerve; jw, jaw; le, first walking leg; nc, nerve cord; pc, protocerebral region; rc, ring commissure; sn, slime papilla nerve; sp; slime papilla.

Figure 3

Structure and position of the adult mouth in the onychophoran Euperipatoides rowelli. Stereomicrographs; anterior is up in all images. (A) Head in ventral view. (B) Head in lateral view. Arrowhead points to the anterior-most pair of lip papillae. (C) Detail of lip papillae (numbered). Note that the mouth opening is encompassed by seven paired papillae (no. 1–7) und one unpaired posterior lip papilla (no. 8). Abbreviations: at, antenna; ey, eye; dp, dermal papillae; jw, jaw; le, first walking leg; lp, lip papillae; ls, lip sensilla; sp, slime papilla; to, tongue; ts, tongue sensilla; vo, ventral organ. Scale bars: 250 μm (A–C).

Figure 4

Morphogenesis of lip papillae encompassing the definitive mouth opening in Euperipatoides rowelli. (A–L) Confocal micrographs of embryonic heads at successive developmental stages in ventrolateral and ventral views. DNA labeling with Bisbenzimide. Developing lip papillae are highlighted by artificial colors. Note that the first pair of lip papillae (number 1 in L and red in F–L) is associated with the first body segment and that it moves from an anterior to a ventral position during development. Note also that the remaining pairs of lip papillae (numbers 2–7 in L) arise lateral to the anlagen of jaws, i.e., in the second body segment, whereas the posterior-most papilla (number 8 in L) originates from a paired anlage in the third body segment (magenta in J–L). Arrowheads in (E–K) point to the developing lip sensilla. Abbreviations: as, anlage of the antennal segment (=cephalic lobe); at, presumptive antenna; dm, definitive mouth opening; ey, eye anlage; ho, anlagen of the hypocerebral organs; jw, developing jaw; le, anlage of the first walking leg; po, embryonic preventral organ of the slime papilla segment; so, openings of the developing salivary glands; sp, developing slime papilla; st, stomodeum; to, embryonic tongue; ve, ventral extraembryonic tissue; vo, developing ventral organ of the slime papilla segment. Scale bars: 250 μm (A–L).

Figure 5

Characteristics of nerves supplying the lip papillae in Euperipatoides rowelli. Maximum projection confocal micrographs of a late stage VII embryo. (A,B) Head in dorsal and ventral views, respectively. Double-labeling with an acetylated α-tubulin antibody (green) and the DNA marker Bisbenzimide (blue). Note the first pair of lip papillae nerves (L1) that originates dorsally and projects ventrally to supply the anterior-most lip papillae (arrowheads). (C,D) Details of the same embryo as in (B). (C) Anti-acetylated α-tubulin immunolabeling (glow mode). Note the dense network of fibers formed by the second (L2) and third lip papillae nerves (L3). (D) The same portion of the embryo as in (C) but mirrored. The three lip papillae nerves (L1, L2, and L3, highlighted by artificial colors) were superimposed on the DNA-labeled surface of the head to illustrate their relationship to the lip papillae. (E) Ventral mouth portion of an embryo in ventral view to demonstrate the origin of the posterior most lip papilla (number 8) from a paired anlage (arrowheads). The lateral lip papillae surrounding the mouth opening are numbered. Note that the paired anlage occupies a position between the ventral and the preventral organs of the slime papilla segment. Phalloidin-rhodamine labeling. Abbreviations: at, antenna; ey, eye; fo, developing frontal organ; jn, jaw nerve; jw, jaw; L1–L3, lip papillae nerves 1–3; le1, first walking leg; po, preventral organ; rc, ring commissure; sn, slime papilla nerve; sp, slime papilla; to, tongue; vo, ventral organ. Scale bars: 100 μm (A–E).

Figure 6

Localization of neuronal somata associated with the first lip papillae nerve in Euperipatoides rowelli. Maximum projection confocal micrographs. Anterior is up in all images. (A) Retrograde fill of the first lip papillae nerve (L1) with dextran coupled to tetramethylrhodamine (glow mode). Note the position of neuronal somata in the posterior portion of the brain, a few additional lateral somata (arrows), and numerous fiber endings that are not associated with any somata (arrowheads). Diagram in the lower right corner illustrates the position of labeled somata and fibers in the brain. (B) Double-fill of the first lip papillae nerve with dextran coupled to fluorescein (green) and the jaw nerve with dextran coupled to tetramethylrhodamine (magenta) from the same body side to reveal the spatial relationship of the corresponding neuronal somata. Note that the somata associated with the first lip papillae nerve are located anterior to those innervating the jaw. Diagram in the lower right corner illustrates the position of labeled somata and fibers in the brain. Abbreviations: dc, deutocerebrum; gr1–gr3; first to third groups of neuronal somata associated with the first lip papillae nerve; jn, jaw nerve; L1, first lip papillae nerve. Scale bars: 100 μm (A,B).

Figure 7

Localization of neuronal somata associated with the second and third lip papillae nerves in Euperipatoides rowelli. Retrograde fills with dextran coupled to a fluorescent marker. Maximum projection confocal micrographs. Anterior is up in all images. Inset illustrates the position of the labeled somata and fibers in the central nervous system. (A) Fill of the second lip papillae nerve (L2). Note the lack of neuronal cell bodies associated with the L2 fibers (arrowheads). (B) Fill of the third lip papillae nerve (L3). Note the position of neuronal cell bodies in the deutocerebrum as well as further posteriorly outside the brain. Abbreviations: ab, anterior bundle; dc, deutocerebrum; al, anterior lateral bundle; am, anterior median bundle; L2 and L3, second and third lip papillae nerves; nc, nerve cord; pb, posterior bundle; pc, protocerebrum; pl, posterior lateral bundle; pm, posterior median bundle; sn, slime papilla nerve. Scale bars: 100 μm (A,B).

Figure 8

Localization of somata of peripheral neurons associated with lip papillae in Euperipatoides rowelli. Anterograde fills with dextran coupled to the fluorescent markers tetramethylrhodamine or fluorescein. Maximum projection confocal micrographs. Lip papillae surrounding the mouth opening are numbered. (A) Overview (external perspective) showing the position of neuronal cell bodies within the lip papillae associated with the second pair of lip papillae nerves (L2). The nerves of each body side were labeled using two different fluorochromes (shown in magenta and green). Note that each nerve supplies the second to fifth pairs of lip papillae. The unpaired eighth papilla is not seen, as it lies beneath the seventh pair of lip papillae. (B) Details of lip papillae from the same preparation as in (A) (anterograde fill of L2; external perspective). Note the bipolar neurons associated with each sensillum (Storch and Ruhberg, 1977). Arrowhead points to a dendrite, arrow to an ascending axon. (C) Overview (internal perspective) showing the position of neuronal somata within the lip papillae associated with the third lip nerve (L3). Note that L3 receives fibers from neuronal cell bodies located in the second to eighth papillae. Corresponding somata within the second papilla are located further externally and, therefore, not seen in this micrograph (but see Supplementary Figure S3). (D) Details of lip papillae from the same preparation as in (B) (anterograde fill of L3; internal perspective). Note the high number of neuronal somata in the proximal portion of each lip papilla. Arrows point to fiber networks. Abbreviations: dm, definitive mouth opening; ls, lip sensillum; L3, third lip papillae nerve, so; soma of a bipolar neuron. Scale bars: 75 μm (A,C) and 50 μm (B,D).

Figure 9

Simplified diagrams summarizing the results of retrograde and anterograde fills of the lip papillae nerves in the onychophoran Euperipatoides rowelli. (A) Position of somata and fibers associated with the three lip papillae nerves, L1, L2, and L3, as revealed by retrograde fills. The innervation pattern of segmental head appendages (antenna, jaw, and slime papilla; see Mayer et al., 2010) is also shown (in gray) to demonstrate their spatial relationship to the lip papillae nerves. (B) Diagram of the onychophoran brain in dorsal view to demonstrate the position of major neural structures and neuropils (combined from various authors, e.g., Schürmann, ; Strausfeld et al., 2006a). (C) Diagram of the onychophoran head in lateral view demonstrating the position of somata and fibers associated with the three nerves (L1, L2, and L3) supplying the lip papillae (numbered from 1 to 8). Based on series of Vibratome sections. (D) Color-coded diagram illustrating the innervation pattern of the lip papillae (numbered) by the corresponding nerves based on anterograde fills. Abbreviations: ag, antennal glomeruli; an, antennal nerve; cb, central body; co, first post-oral commissure; cp, connecting piece; dc, deutocerebral brain region; dm; definitive mouth opening; ey, eye; jn, jaw nerve; L1–L3, lip papillae nerves 1–3; le, first walking leg; ln, anterior and posterior nerves of the first walking leg; mb, lateral lobes of mushroom body; nc, nerve cord; on, optic nerve; pc, protocerebral brain region; sn, slime papilla nerve; sr, region of the nerve central nervous system supplying the slime papillae.